Insertion of an Arginine Residue into the Transmembrane Segments Corrects Protein Misfolding

Abstract

Deletion of Phe-508 (⌬F508) in cystic fibrosis transmembrane conductance regulator causes cystic fibrosis because of misfolding of the protein. P-glycoprotein (P-gp) containing the equivalent mutation (⌬Y490) is also misfolded but can be rescued with drug substrates. Whether rescue is due to direct binding of drug substrate to the transmembrane (TM) segments or to indirect effects on cellular protein folding pathways is still controversial. P-gpdrug substrate interactions likely involve hydrogen bonds. If the mechanism of drug rescue involves changes to TM packing then we should be able to identify suppressor mutations in the TM segments that can mimic the drug rescue effects. We predicted that an arginine residue in the TM segments predicted to line the drugbinding pocket of P-gp (I306(TM5) or F343(TM6)) might suppress ⌬Y490 P-gp protein misfolding because it has the highest propensity to form hydrogen bonds. We show that R306(TM5) or R343(TM6) increased the relative amount of mature ⌬Y490 P-gp by 6-fold. Most other changes to Ile-306 or Phe-343 did not enhance maturation of ⌬Y490 P-gp. The I306R mutant also promoted maturation of misprocessed mutants that had mutations in the second nucleotide-binding domain (L1260A), the cytoplasmic loops (G251V, F804A), the linker region (P709A), or in TM segments (G300V, G722A). These results show that arginine residues in the TM domains can mimic the drug rescue effects and are effective suppressor mutations for processing mutations located throughout the molecule.Cystic Fibrosis (CF) 2 is a lethal inherited disorder caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein (1). The most common CF-associated mutation is deletion of Phe508 (⌬F508) in the first nucleotide-binding domain (NBD1) (2). The mutation causes protein misfolding. The misfolded protein is retained in the endoplasmic reticulum and is rapidly degraded (3, 4). The equivalent deletion in human P-glycoprotein (⌬Y490) also leads to defective folding and rapid degradation (5).The presence of the ⌬F508 mutation in CFTR has little effect on the conformation of NBD1, its in vitro stability, or folding kinetics (6). The primary effect of ⌬F508 is disruption of NBD1-TMD1 interactions. In P-gp, the ⌬Y490 mutation also disrupts NBD1-TMD1 interactions (7). Disruption of NBD1-TMD1 interactions then appears to disrupt subsequent interactions between TMD1 and TMD2 (8,9).An important clinical goal in CF is to specifically correct the folding defect in ⌬F508-CFTR as the mature protein still has cAMP-activated chloride channel activity (10). Studies on the ⌬Y490 P-gp model system suggest that specific drug interactions with the TM segments can promote maturation of the mutant protein. Therefore, the results imply that the TM segments of ⌬F508-CFTR would be a good target for the development of pharmacological chaperones. The mechanism of how pharmacological chaperones rescue folding defects in P-gp (11) is controversial because it is difficult to distinguish whether i...

Arginines in the First Transmembrane Segment Promote Maturation of a P-glycoprotein Processing Mutant by Hydrogen Bond Interactions with Tyrosines in Transmembrane Segment 11

“…3 shows that the 150-kDa immature protein was sensitive to both endoglycosidases, whereas the 170-kDa protein was only sensitive to endoglycosidase F. These results indicate that the 150-kDa immature proteins were only core-glycosylated, whereas the 170-kDa mature proteins were processed in the Golgi. Cell-surface labeling studies showed that only the mature 170-kDa protein was labeled (data not shown) and confirmed the previous observation that the 170-kDa protein was at the cell surface (8,9). Similar results were obtained with the other arginine mutants (data not shown).…”

“…Processing mutations appear to inhibit folding by disrupting packing of TM segments at the interface between the TMDs (6), a defect that can be repaired by expression in the presence of drug substrates (6,7). Drug substrates appear to rescue P-gp processing mutants through direct interactions with the TM segments because their effects can be mimicked by introducing arginine mutations into positions predicted to line the drug binding domain such as position 65 in TM segment 1 (8,9). It was found that the L65R mutation promoted maturation of P-gp processing mutants such as ⌬Y490 and G251V (9).…”

A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

“…Better understanding of how Mdr transporters respond conformationally to dissimilar substrates in a transport-competent manner would add mechanistically important clues to how dissimilar compounds are actively exported by such transporters. For example, in a P-glycoprotein mutant that is defective in biogenesis, it has been reported that mutations in the binding site improve protein maturation and that this effect can also be achieved by substrates (27)(28)(29)(30)(31)(32). Thus, binding site modifications also have substrate-mimicking effects in this ATP-binding cassette Mdr transporter.…”

Suppressor Mutations in the Transmembrane Segments of P-glycoprotein Promote Maturation of Processing Mutants and Disrupt a Subset of Drug-binding Sites

“…Therefore, changes to the TM5-TM6 hairpin can affect distal folding events. Some changes in TM5 (I306A or G) or in TM6 (F343Y) in the ⌬Y490 P-gp mutant also caused the mutant protein to be unstable because cells expressing these mutants contained relatively large amounts of a 130-kDa degradation product (21). An example of the effects of Arg and Tyr changes to Phe 343 in mutant ⌬Y490 P-gp is shown in Fig.…”

“…essing mutants by a direct mechanism because suppressor mutations (arginines) introduced into TM segments 5 (I306R) and 6 (F343R) also promoted maturation of the protein (21). One explanation for this observation was that the arginine residues mimicked the effects of drug substrates by promoting interactions among the TM segments.…”